The overall objective of the proposed research is to identify and characterize mechanisms responsible for intestinal transport of bile acids. To accomplish this objective, the first specific aim is to identify bile acid binding polypeptides in ileal basolateral membranes that are involved in bile acid transport using photoaffinity labeling. Studies are proposed to demonstrate that photolabile bile acid derivatives interact with and reversibly and irreversibly inhibit the recently described bile acid transport system in the basolateral membrane. Photoaffinity labeling of basolateral membranes with these derivatives then are performed to identify bile acid binding polypeptides using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Binding polypeptides involved in bile acid transport in ileal basolateral membrane vesicles will be compared to those in jejunal plasma membranes, ileal brush border membranes and plasma membranes of hepatocytes. The second specific, yet inter-related, aim is to identify the intracellular pathway of bile acid transport. From a description of the kinetics and characteristics of bile acid uptake (or lack of uptake) by epithelial cell organelles a model of transcellular transport can be proposed. Photoaffinity labeling with bile acid photoprobes of intact enterocytes and subsequent cellular fractionation should identify subcellular sites of bile acid covalent binding. Pulse-chase experiments are desinged to follow the transepithelial route of bile acids with respect to time. The methods of procedure combine the sequential breakdown of the small intestine with covalent binding techniques to identify subcellular components that are intimately involved in bile acid transport. These experiments should provide not only greater understanding of the controlling mechanisms for bile acid intestinal transport but also insight into the basic but unresolved question of how transepithelial translocation occurs for all transport systems.